Abstract

Embryos and larvae of sea urchins (Lytechinus variegatus, Strongylocentrotus droebachiensis, Strongylocentrotus purpuratus, Dendraster excentricus), and starfish (Pisaster ochraceus) were investigated for the presence of a functional endocannabinoid system. Anandamide (arachidonoyl ethanolamide, AEA), was measured in early L. variegatus embryos by liquid chromatography/mass spectrometry. AEA showed a strong developmental dynamic, increasing more than 5-fold between the 8-16 cell and mid-blastula 2 stage. 'Perturb-and-rescue' experiments in different sea urchin species and starfish showed that AEA blocked transition of embryos from the blastula to the gastrula stage, but had no effect on cleavage divisions, even at high doses. The non-selective cannabinoid receptor agonist, CP55940, had similar effects, but unlike AEA, also blocked cleavage divisions. CB1 antagonists, AEA transport inhibitors, and the cation channel transient membrane potential receptor V1 (TrpV1) agonist, arachidonoyl vanillic acid (arvanil), as well as arachidonoyl serotonin and dopamine (AA-5-HT, AA-DA) acted as rescue substances, partially or totally preventing abnormal embryonic phenotypes elicited by AEA or CP55940. Radioligand binding of [(3)H]CP55940 to membrane preparations from embryos/larvae failed to show significant binding, consistent with the lack of CB receptor orthologs in the sea urchin genome. However, when binding was conducted on whole cell lysates, a small amount of [(3)H]CP55940 binding was observed at the pluteus stage that was displaced by the CB2 antagonist, SR144528. Since AEA is known to bind with high affinity to TrpV1 and to certain G-protein-coupled receptors (GPCRs), the ability of arvanil, AA-5-HT and AA-DA to rescue embryos from AEA teratogenesis suggests that in sea urchins AEA and other endocannabinoids may utilize both Trp and GPCR orthologs. This possibility was explored using bioinformatic and phylogenetic tools to identify candidate orthologs in the S. purpuratus sea urchin genome. Candidate TrpA1 and TrpV1 orthologs were identified. The TrpA1 ortholog fell within a monophyletic clade, including both vertebrate and invertebrate orthologs, whereas the TrpV1 orthologs fell within two distinct TrpV-like invertebrate clades. One of the sea urchin TrpV orthologs was more closely related to the vertebrate epithelial calcium channels (TrpV5-6 family) than to the vertebrate TrpV1-4 family, as determined using profile-hidden Markov model (HMM) searches. Candidate dopamine and adrenergic GPCR orthologs were identified in the sea urchin genome, but no cannabinoid GPCRs were found, consistent with earlier studies. Candidate dopamine D(1), D(2) or alpha(1)-adrenergic receptor orthologs were identified as potential progenitors to the vertebrate cannabinoid receptors using HMM searches, depending on whether the multiple sequence alignment of CB receptor sequences consisted only of urochordate and cephalochordate sequences or also included vertebrate sequences.

Detection and measurement of AEA by LC/MS in L. variegatus embryos. Ion chromatograms of AEA and AEA-d8 in A 8–16 cell stage and B mid-blastula 2 stage embryos. AEA was quantified using AEA-d8 as internal standard, where 90 pmol was added to the sea urchin samples. The amounts of AEA measured at the 8–16 cell and mid-blastula stage were 1.33 ± 0.12 and 6.8 ± 3.5 pmol/g, respectively.

AEA perturbs the movement of PMC shown in green, during gastrulation. A Vehicle control; B AEA 25 μM; C vanilloid receptor agonist, arvanil 40 μM, partially prevented the effect of AEA, such that the ring of PMC at the vegetal pole is clearly visible in both A and C (asterisk). Embryos stained with PMC marker 1D5 [].

Developmental dynamics of sensitivity to CB2 receptor antagonist, AM-630 and AEA. Substances were given at the 2– 4 cell stage (B–E) or at mid-blastula 2 (G–I). Controls (A, F). AM-630 blocked cleavage divisions as shown by embryos composed of multiple blastomeres in B. AEA acted as a rescue substance to unblock cleavage divisions, as shown in C. However, when given at mid-blastula 2, AM-630 partially rescued from the effects of AEA (H). A–E Imaged at mid-blastula 2; F–H imaged at late gastrula. Scale bar = 50 μm.

Phylogenetic relationships of Trp and GPCRs in sea urchins, other invertebrates and vertebrates. A Phylogenetic tree of TrpA and TrpV family receptors. Invertebrate and vertebrate species contained a single TrpA gene that formed a monophyletic clade (a). Invertebrate TrpV orthologs fell into 2 families, one that contained the worm (C. elegans) OSM-9, fruitfly (D. melanogaster) inactive (b), while the other contained the family of worm OCR proteins, fruitfly nanchung, and sea squirt nanLIKE (c). Vertebrate TrpV orthologs also fell into two families, the epithelial calcium channels (ECaC; TrpV5-6) (d) and the TrpV1-like channels (TrpV1-4) (e). Profile HMM searches against the sea urchin genome, with multiple sequence alignments (MSA) containing vertebrate TrpV family members as the query, indicated that the closest similarity between vertebrate and invertebrate TrpV subunits was between the vertebrate epithelial calcium channels (ECaC; TrpV5-6) (d) and the invertebrate sea urchin (S. purpuratus) GLEAN3 06793 in the OCR and nanchung clade (c). GLEAN3 09599 was also identified as a homolog to the vertebrate TrpV proteins in the profile HMM searches, with E-values of 1.0−7 and 1.8−3 in the TrpV5-6 and TrpV1-4 searches, respectively. The TrpA1 ortholog in sea urchins, GLEAN3 15403, had E-values higher than 1.5−4 in the two HMM searches. B Phylogenetic tree of GPCRs. Cannabinoid, Edg-like lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) receptors were found only in vertebrates and the urochordates sea squirt (C. intestinalis) and Pacific sea human and fruitfly D1-5 dopamine receptors formed a clade with multiple sea urchin GLEAN3 sequences, including the adrenergic-like GLEAN3 21588 and D1-like GLEAN3 11320 (h). HMM searches against the sea urchin genome including all currently identified GPCR receptors, using the urochordate and cephalochordate CB receptors as query, identified the sea urchin GLEAN3 21588 as the most similar. HMM searches with vertebrate CB sequences also included in the search alignment identified the D1-like GLEAN3 11320 as the most similar. All other GLEAN3 GPCR receptors in the phylogenetic tree had bit scores lower than −55.5 and E-values higher than 3.0−7 for the first HMM search containing only the urochordate and cephalochordate sequences. In the search also including vertebrate CB receptors, all other GLEAN3 GPCR receptors in the phylogenetic tree had bit scores lower than −43.1 and E-values higher than 1.5−10.